Due to its unique chemical properties iron has immense versatility in biology. Although this metal is used for myriad of critical processes, in an aerobic environment this comes at a potentially high cost to cells in the form of oxidative damage to proteins, DNA/RNA, and lipids. Mammals have therefore evolved complex regulatory mechanisms devoted to iron homeostasis, where iron regulatory protein 2 (IRP2) serves a critical function. IRP2 is an RNA-binding protein that regulates mRNAs encoding proteins important in iron storage (ferritin) and iron uptake (transferrin receptor). The coupling of cellular iron with the appropriate regulation of these, and other mRNAs, is achieved through iron and oxygen stimulation of IRP2 ubiquitination/degradation. Defining the molecular mechanisms by which IRP2 senses physiologic changes in iron and oxygen and how this signals for its degradation is essential for a comprehensive understanding of mammalian iron homeostasis. Towards this end, we have recently identified a putative iron-binding motif in IRP2. We hypothesize that this site confers a novel iron- and oxygen-dependent enzymatic activity to IRP2, and that this activity is involved in its degradation through a self-modifying mechanism or in catalyzing modifications on other substrates. The specific aims addressed in this R21 application are: (1) Characterize iron-binding to the putative iron-binding site on IRP2 and examine its potential role in mediating IRP2 degradation or in direct regulation of IRP2 target mRNAs and (2) Examine novel iron-dependent IRP2 enzymatic activity. The goal of this work is to define hitherto unidentified IRP2 biochemical activity and identify pathways in which it participates. This work is presented as an R21 application since it satisfies the aims of the R21 mechanism by representing an exploratory high risk study with potentially high impact.